Refolding Record:

Protein See all refolding records for this protein...
Protein Name	Vascular endothelial growth factor
Abbreviated Name	VEGF
SCOP Family	Platelet-derived growth factor-like
Structure Notes
Organism	Human
UniProt Accession	P15692
SCOP Unique ID	n/a
Structure Solved
Class	Small Proteins
Molecularity	Unknown

Construct
Full Length	n
Domain	n/a
Chimera	n/a
Variants	VEGF121 isoform
Chain Length	127
Molecular Weight	14940.0
Pi	6.28762
Molecular Weight	14940.0
Disulphides	Unknown
Full Sequence	MHHHHHH PMA EGGGQNHHEV VKFMDVYQRS YCHPIETLVD IFQEYPDEIE YIFKPSCVPL MRCGGCCNDE GLECVPTEES NITMQIMRIK PHQGQHIGEM SFLQHNKCEC RPKKDRARQE NCDKPRR
Notes	n/a

Expression
Report	Siemeister G, Schnurr B, Mohrs K, Schachtele C, Marme D, Martiny-Baron G (1996) Biochemical and Biophysical Research Com, 222, 249-255
Project Aim	Drug Studies
Fusion	N-terminal hexahistidine tag
Protein Expression and Production	Protein recombinantly expressed as and refolded from inclusion bodies.
Expression Host	Escherichia coli
Expression Strain	BL21(DE3)
Expression Temp	37.0
Expression Time	3h
Expression Vector	pET3d
Expression Protocol	Cells were grown in 250ml LB medium containing 100microg/ml ampicillin at 37degC. When OD600 reached 0.8, IPTG was added to a final concentration of 0.4mM and the culture was grown for another 3h.
Method of Induction	IPTG
Cell Density at Induction	OD 600 = 0.8
Cell Disruption Method	Freeze/Thaw+Sonication
Lytic Agent	Lysozyme
Pre-Refolding Purification	None
Solubility	insoluble

Refolding
Refolding Method	Dilution
Wash Buffer	50mM TrisHCl, 10mM 2-mercaptoethanol, 2mM EDTA, 5%(v/v) glycerol, 0.2mg/ml lysozyme, 10microg/ml DNaseI, 0.05% Na-deoxycholate, 1% NP-40 pH 8.0
Solubilization Buffer	6M urea, 0.1M DTT, 50mM MES pH 5.5
Refolding Buffer	PBS, 50mM glycine
Pre-Refolding Purification	None
Tag Cleaved	no
Refolding pH	7.4
Refolding Temperature	4.0
Protein Concentration
Refolding Time
Redox Agent	None
Redox Agent Concentration	n/a
Refolding Protocol	Cells were harvested by centrifugation (10min, 5000rpm, 4degC) and the pellet was resuspended in 2-ml of 20mM TrisHCl, 150mM NaCl pH 7.5. The suspension was centrifuged again (10min, 5000rpm, 4degC) and the pellet was then frozen at -80degC, thawed at 37degC and resuspdended in 25ml wash buffer without Na-deoxycholate or NP-40 and incubated for 30min at 22degC. The suspension was sheared by five high-speed treatments of 20s in an Ultra-Turrax dispersing apparatus and incubated for 10min at 22degC. The mixture was cooled on ice and sonicated six times (15s). After addition of Na-deoxycholate (0.05%) and NP-40(1%(w/v)), the mixture was incubated for 10min at 4degC and then centrifuged (10000rpm, 4degC, 30min). The pellet was then resuspended in 25ml wash buffer and recentrifuged. The inclusion body pellet was then resuspended in 25ml solubilization buffer at 4degC. The solubilized protein was then concentrated by ultrafiltration and refolded by sequential dilution to a final concentration of 0.3M urea by the addition of PBS/50mM Glycine. The refefolded protein was then purified by Ni2+ chelating affinity chromatography on a 1mL Ni-NTA resin, followed by buffer exchange by ultrafiltration to 0.1 N acetic acid
Refolding Assay	SDS-PAGE
Refolding Chaperones	None
Refolding Additives	None,Glycine
Additives Concentration	50mM
Refolding Yield	5-10mg/L culture
Purity	95%
Notes	Similar procedure also for VEGF165 reported in the same paper

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